Project Summary Pathogenic bacteria display polymeric virulence factors to establish and maintain infections. We will investigate the mechanisms through which these polymers are produced and search for small molecule polymer assembly inhibitors. Two types of surface polymers in Gram-positive bacteria will be studied, (i) pili, proteinaceous fibers that project from the cell surface to mediate adhesion to host tissues, and (ii) Wall Teichoic Acids (WTAs), highly abundant glycopolymers that play a fundamental role in maintaining the integrity of the cell wall. Both pili and WTA polymers are important virulence factors, but how they are synthesized is poorly understood. In aims #1- 2, we will study how the archetypal SpaA-pilus from Corynebacterium diphtheria is assembled by sortase polymerases. These enzymes catalyze a unique transpeptidation reaction that covalently links adjacent protein pilus subunits together via a lysine isopeptide bond, thereby conferring enormous tensile strength that enables bacterial adherence. By synergistically employing structural, biochemical, cellular and chemical tools, we will learn how sortase polymerases build the pilus shaft and define the structure of the fundamental building block from which all Gram-positive pili are constructed. We will also determine the molecular basis through which pilus biogenesis is terminated via a novel handoff mechanism in which the pilus is transferred between tandemly arranged sortases on the cell surface. This work will have a broad impact, as a wide range of pathogenic microbes use a similar mechanism to assemble their pili. In aim #3, we will study how Gram-positive bacteria produce WTA using the TarA enzyme, a novel glycosyltransferase that catalyzes the first committed step in polymer biosynthesis. TarA is a promising drug target, as clinically relevant methicillin-resistant Staphylococcus aureus (MRSA) is defective in host colonization and re-sensitized to Beta-lactam drugs when WTA biosynthesis is disrupted. Crystal structures of TarA in its apo- and substrate-bound forms will be determined, facilitating the rational exploration of its catalytic mechanism. High-throughput screening using a novel, cell-based bacterial cytological profiling assay will also be performed to discover small molecule TarA inhibitors that could have useful therapeutic properties. Combined, studies of pilus and WTA biogenesis will provide fundamental insight into the chemistry underlying polymer assembly in Gram-positive bacteria and could lead to novel antibiotics to treat infections caused by MRSA and other multi-drug resistant bacteria.